The overall direction of the Molecular Mechanisms of Tumor Promotion Section Is to understand the mechanism of action of the phorbol esters and their endogenous analog, sn-1,2-diacylglycerol. Particular emphasis Is directed at clarifying the basis for heterogeneity of response of this system to Its various ligands. In collaboratIon with several chemistry groups, we are extending our knowledge of the stereospecific requirements for binding and for activity. This effort has generated high affinity synthetic ligands for protein kinase C (PKC), the primary receptor for the phorbol esters, as well as identified additional classes of ligands with moderate (microM) affinity. We have examined the phorbol ester structure activity relations both for Individual Isozyme of PKC and for their Isolated binding domains (zinc finger regions). We demonstrate that those derivatives which function as partial antagonists, e.g. bryostatin 1 and prostrating, induce differential down regulation of PKC In Intact cells. By reconstitution and overexpression, we find that different PKC isozyme fulfill different biochemical functions. We have characterized two classes of high affinity phorbol ester receptors distinct from PKC, viz. n-chimaerin and unc-13. N-chimaerin shares with PKCalpha close similarity in structure activity relations, in lipid dependence, and In sensitivity to inhibitors targeted to the regulatory domain of PKC. Although the analysis for unc-13 Is not complete, so far a similar situation prevails finally, we continue to characterize the receptors for resiniferatoxin, a related class of phorbol related diterpenes which act as ultrapotent vanilloid (capsaicin) analogs. Binding shows marked positive cooperativity; human receptors are similar to those from rats, albeit with modestly weaker affinity.